Functional and structural characterization of rhodopsin oligomers

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-beta-D-maltoside, n-tetradecyl-beta-D-maltoside, and n-hexadecyl-beta-D-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-beta-D-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-beta-D-maltoside and n-hexadecyl-beta-D-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-beta-D-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.     

The role of Glu181 in the photoactivation of rhodopsin

The visual pigment rhodopsin is a prototypical seven transmembrane helical G protein-coupled receptor. Photoisomerization of its protonated Schiff base (PSB) retinylidene chromophore initiates a progression of metastable intermediates. We studied the structural dynamics of receptor activation by FTIR spectroscopy of recombinant pigments. Formation of the active state, Meta II, is characterized by neutralization of the PSB and its counterion Glu113. We focused on testing the hypothesis of a PSB counterion switch from Glu113 to Glu181 during the transition of rhodopsin to the still inactive Meta I photointermediate. Our results, especially from studies of the E181Q mutant, support the view that both Glu113 and Glu181 are deprotonated, forming a complex counterion to the PSB in rhodopsin, and that the function of the primary counterion shifts from Glu113 to Glu181 during the transition to Meta I. The Meta I conformation in the E181Q mutant is less constrained compared with that of wild-type Meta I. In particular, the hydrogen bonded network linking transmembrane helices 1, 2, and 7, adopts a conformation that is already Meta II-like, while other parts of the receptor appear to be in a Meta I-like conformation similar to wild-type. We conclude that Glu181 is responsible, in part, for controlling the extraordinary high pK(a) of the chromophore PSB in the dark state, which very likely decreases upon transition to Meta I in a stepwise weakening of the interaction between PSB and its complex counterion during the course of receptor activation. A model for the specific role in coupling chromophore isomerization to protein conformational changes concomitant with receptor activation is presented.     

Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface

Although homo- and heterodimerizations of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers are able to assemble in different ways, thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon, which remain to be clarified, could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full-length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4, and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column toward smaller molecular masses with a reduced propensity for dimer formation in a cross-linking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay from which dissociation constants were calculated. A BRET (bioluminescence resonance energy transfer) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Although disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of G_t to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking.     

Measurement of Slow Spontaneous Release of 11-cis-Retinal from Rhodopsin

The vertebrate visual photoreceptor rhodopsin (Rho) is a unique G protein-coupled receptor as it utilizes a covalently tethered inverse agonist (11-cis-retinal) as the native ligand. Previously, electrophysiological studies showed that ligand binding of 11-cis-retinal in dark-adapted Rho was essentially irreversible with a half-life estimated to be 420 years, until after thermal isomerization to all-trans-retinal, which then slowly dissociates. This long lifetime of 11-cis-retinal binding was considered to be physiologically important for minimizing background signal (dark noise) of the visual system. However, in vitro biochemical studies on the thermal stability of Rho showed that Rho decays with a half-life on the order of days. In this study, we resolve the discrepancy by measuring the chromophore exchange rate of the bound 11-cis-retinal chromophore with free 9-cis-retinal from Rho in an in vitro phospholipid/detergent bicelle system. We conclude that the thermal decay of Rho primarily proceeds through spontaneous breaking of the covalent linkage between opsin and 11-cis-retinal, which was overlooked in the electrophysiological recording. We estimate that this slow spontaneous release of 11-cis-retinal from Rho should result in 10⁴ to 10⁵ free opsin molecules in a dark-adapted rod cell—a number that is three orders of magnitude higher than previously expected. We also discuss the physiological implications of these findings on the basal activity of opsins and the associated dark noise in the visual system.     

Biochemical and physiological properties of rhodopsin regenerated with 11-cis-6-ring- and 7-ring-retinals

Phototransduction is initiated by the photoisomerization of rhodopsin (Rho) chromophore 11-cis-retinylidene to all-trans-retinylidene. Here, using Rho regenerated with retinal analogs with different ring sizes, which prevent isomerization around the C(11)=C(12) double bond, the activation mechanism of this G-protein-coupled receptor was investigated. We demonstrate that 11-cis-7-ring-Rho does not activate G-protein in vivo and in vitro, and that it does not isomerize along other double bonds, suggesting that it fits tightly into the binding site of opsin. In contrast, bleaching 11-cis-6-ring-Rho modestly activates phototransduction in vivo and at low pH in vitro. These results reveal that partial activation is caused by isomerization along other double bonds in more rigid 6-locked retinal isomers and protonation of key residues by lowering pH in 11-cis-6-ring-Rhos. Full activation is not achieved, because isomerization does not induce a complete set of conformational rearrangements of Rho. These results with 6- and 7-ring-constrained retinoids provide new insights into Rho activation and suggest a potential use of locked retinals, particularly 11-cis-7-ring-retinal, to inactivate opsin in some retinal degeneration diseases.     

Conformational changes in the photocycle of Anabaena sensory rhodopsin: absence of the Schiff base counterion protonation signal

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.     

Mechanism of rhodopsin activation as examined with ring-constrained retinal analogs and the crystal structure of the ground state protein

The guanine nucleotide-binding protein (G-protein)-coupled receptor superfamily (GPCR) is comprised of a large group of membrane proteins involved in a wide range of physiological signaling processes. The functional switch from a quiescent to an active conformation is at the heart of GPCR action. The GPCR rhodopsin has been studied extensively because of its key role in scotopic vision. The ground state chromophore, 11-cis-retinal, holds the transmembrane region of the protein in the inactive conformation. Light induces cis-trans isomerization and rhodopsin activation. Here we show that rhodopsin regenerated with a ring-constrained 11-cis-retinal analog undergoes photoisomerization; however, it remains marginally active because isomerization occurs without the chromophore-induced conformational change of the opsin moiety. Modeling the locked chromophore analogs in the active site of rhodopsin suggests that the beta-ionone ring rotates but is largely confined within the binding site of the natural 11-cis-retinal chromophore. This constraint is a result of the geometry of the stable 11-cis-locked configuration of the chromophore analogs. These results suggest that the native chromophore cis-trans isomerization is merely a mechanism for repositioning of the beta-ionone ring which ultimately leads to helix movements and determines receptor activation.     

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.     

Structure of bacteriorhodopsin at 1.55 A resolution.

Th?e atomic structure of the light-driven ion pump bacteriorhodopsin and the surrounding lipid matrix was determined by X-ray diffraction of crystals grown in cubic lipid phase. In the extracellular region, an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base and the proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline? kink. The bulge is stabilized by hydrogen-bonding of the main-chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The results indicate extensive involvement of bound water molecules in both the structure and the function of this seven-helical membrane protein. A bilayer of 18 tightly bound lipid chains forms an annulus around the protein in the crystal. Contacts between the trimers in the membrane plane are mediated almost exclusively by lipids.     

A naturally occurring mutation of the opsin gene (T4R) in dogs affects glycosylation and stability of the G protein-coupled receptor

Rho (rhodopsin; opsin plus 11-cis-retinal) is a prototypical G protein-coupled receptor responsible for the capture of a photon in retinal photoreceptor cells. A large number of mutations in the opsin gene associated with autosomal dominant retinitis pigmentosa have been identified. The naturally occurring T4R opsin mutation in the English mastiff dog leads to a progressive retinal degeneration that closely resembles human retinitis pigmentosa caused by the T4K mutation in the opsin gene. Using genetic approaches and biochemical assays, we explored the properties of the T4R mutant protein. Employing immunoaffinity-purified Rho from affected RHO(T4R/T4R) dog retina, we found that the mutation abolished glycosylation at Asn(2), whereas glycosylation at Asn(15) was unaffected, and the mutant opsin localized normally to the rod outer segments. Moreover, we found that T4R Rho(*) lost its chromophore faster as measured by the decay of meta-rhodopsin II and that it was less resistant to heat denaturation. Detergent-solubilized T4R opsin regenerated poorly and interacted abnormally with the G protein transducin (G(t)). Structurally, the mutation affected mainly the "plug" at the intradiscal (extracellular) side of Rho, which is possibly responsible for protecting the chromophore from the access of bulk water. The T4R mutation may represent a novel molecular mechanism of degeneration where the unliganded form of the mutant opsin exerts a detrimental effect by losing its structural integrity.     

Potent activation of RhoA by Galpha q and Gq-coupled receptors

Heterotrimeric G proteins of the G(i), G(s), and G(q) family control a wide array of physiological functions primarily by regulating the activity of key intracellular second messenger-generating systems. alpha subunits of the G(12) family, Galpha(12) and Galpha(13), however, can promote cellular responses that are independent of conventional second messengers but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Galpha(12/13) and Rho stimulation. Recent observations suggest that many cellular responses elicited by Galpha(q) and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Galpha(q) may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Galpha(q) and its coupled receptors can stimulate endogenous Rho. Here we show that the expression of activated forms of Galpha(q) and the stimulation of G(q)-coupled receptors or chimeric Galpha(q) molecules that respond to G(i)-linked receptors can promote a robust activation of endogenous Rho in HEK-293T cells. Interestingly, this response was not prevented by molecules interfering with the ability of Galpha(13) to stimulate its linked RhoGEFs, together suggesting the existence of a novel molecular mechanism by which Galpha(q) and the large family of G(q)-coupled receptors can regulate the activity of Rho and its downstream signaling pathways.     

Transducer binding establishes localized interactions to tune sensory rhodopsin II

In haloarchaea, sensory rhodopsin II (SRII) mediates a photophobic response to avoid photo-oxidative damage in bright light. Upon light activation the receptor undergoes a conformational change that activates a tightly bound transducer molecule (HtrII), which in turn by a chain of homologous reactions transmits the signal to the chemotactic eubacterial two-component system. Here, using single-molecule force spectroscopy, we localize and quantify changes to the intramolecular interactions within SRII of Natronomonas pharaonis (NpSRII) upon NpHtrII binding. Transducer binding affected the interactions at transmembrane alpha helices F and G of NpSRII to which the transducer was in contact. Remarkably, the interactions were distributed asymmetrically and significantly stabilized alpha helix G entirely but alpha helix F only at its extracellular tip. These findings provide unique insights into molecular mechanisms that "prime" the complex for signaling, and guide the receptor toward transmitting light-activated structural changes to its cognate transducer.     

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ～40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.     

Normal Activation of Discoidin Domain Receptor 1 Mutants with Disulfide Cross-links,Insertions, or Deletions in the Extracellular Juxtamembrane Region: MECHANISTIC IMPLICATIONS*

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters.     

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.     

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β₂-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β₂-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.     

Helix 8 plays a crucial role in bradykinin B(2) receptor trafficking and signaling

Upon activation the human bradykinin B(2) receptor (B(2)R) acts as guanine nucleotide exchange factor for the G proteins G(q/11) and G(i). Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits β-arrestins, which block further G protein activation and promote B(2)R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B(2)R. We show here that disruption of helix 8 in the B(2)R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helix-breaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B(2)R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B(2)R with GRK2/3 and β-arrestin2 as shown by co-immunoprecipitation. Recruitment of β-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B(2)R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B(2)R with GRK2/3 and β-arrestins. Additional data obtained with chimera of B(2)R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well.     

Rhodopsin and 9-demethyl-retinal analog: effect of a partial agonist on displacement of transmembrane helix 6 in class A G protein-coupled receptors

Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis-->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl-retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling. Earlier site-directed spin labeling studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of [corrected] transmembrane helix 6 (TM6) by approximately 6A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or [corrected] by anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm pigments. When the proton concentration is further increased, a new state arises in 9-dm pigments that is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state.     

Crystal structure of rhodopsin: implications for vision and beyond

A heptahelical transmembrane bundle is a common structural feature of G-protein-coupled receptors (GPCRs) and bacterial retinal-binding proteins, two functionally distinct groups of membrane proteins. Rhodopsin, a photoreceptor protein involved in photopic (rod) vision, is a prototypical GPCR that contains 11-cis-retinal as its intrinsic chromophore ligand. Therefore, uniquely, rhodopsin is a GPCR and also a retinal-binding protein, but is not found in bacteria. Rhodopsin functions as a typical GPCR in processes that are triggered by light and photoisomerization of its ligand. Bacteriorhodopsin is a light-driven proton pump with an all-trans-retinal chromophore that photoisomerizes to 13-cis-retinal. The recent crystal structure determination of bovine rhodopsin revealed a structure that is not similar to previously established bacteriorhodopsin structures. Both groups of proteins have a heptahelical transmembrane bundle structure, but the helices are arranged differently. The activation of rhodopsin involves rapid cis-trans photoisomerization of the chromophore, followed by slower and incompletely defined structural rearrangements. For rhodopsin and related receptors, a common mechanism is predicted for the formation of an active state intermediate that is capable of interacting with G proteins.     

Differential regulation of Rho and Rac through heterotrimeric G-proteins and cyclic nucleotides

Platelets were used to study the activation of Rho and Rac through G-protein-coupled receptors and its regulation by cyclic nucleotides. The thromboxane A(2) (TXA(2)) mimetic rapidly activated both small GTPases independently of integrin alpha(IIb)beta(3) activation., which leads to the activation of G(12)/G(13) and G(q) did not induce Rac activation in G alpha(q)-deficient platelets but was able to activate Rho, to stimulate actin polymerization and phosphatidylinositol 4,5-bisphosphate formation, and to induce shape change. Rac activation by in wild-type platelets could be blocked by chelation of intracellular Ca(2+) and was partially sensitive to apyrase and AR-C69931MX, an antagonist of the G(i)-coupled ADP receptor. Cyclic AMP, which completely blocks platelet function, inhibited the -induced activation of G(q) and G(12)/G(13) as well as of Rac and Rho. In contrast, cGMP, which has no effect on platelet shape change blocked only activation of G(q) and Rac. These data demonstrate that Rho and Rac are differentially regulated through heterotrimeric G-proteins. The G(12)/G(13)-mediated Rho activation is involved in the shape change response, whereas Rac is activated through G(q) and is not required for shape change. Cyclic AMP and cGMP differentially interfere with -induced Rho and Rac activation at least in part by selective effects on the regulation of individual G-proteins through the TXA(2) receptor.